Excess lipid accumulation in non-adipose tissues is associated with cellular dysfunction and cell death that may contribute to the pathogenesis of insulin resistance, non-alcoholic steatohepatitis, and cardiomyopathy in diabetes and obesity. Mechanisms involved in these pathophysiologic responses have been studied in cultured cells by supplementation of growth media with high concentrations of free fatty acids. The saturated fatty acid palmitate leads to apoptosis in cultured cells by a mechanism involving oxidative stress and initiation of ER stress. This proposal will test the hypothesis that fatty acid perturbation of normal endoplasmic reticulum function is an early event in this lipotoxic cell death. Moreover, cells with gene disruptions that prevent lipotoxic cell death may have defects in fatty acid import and channeling or in the response to lipid-induced perturbations of endoplasmic reticulum function or oxidative stress. Our first three aims will employ biochemical and genetic approaches to identify molecular targets and signaling pathways in the lipotoxic response, In our fourth aim, we will translate our findings to mouse models relevant to diabetic cardiovascular disease in which lipid accumulation in cardiomyocytes is associated with heart failure. The results of these studies will characterize fundamental aspects of cellular lipid homeostasis. Moreover, these studies will provide new insights into the lipotoxic response to excess lipid accumulation in non-adipose tissues in diabetes and obesity.